Abstract

Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.

Notes

Acknowledgements

The LeGO lentiviral vectors containing the human reprogramming transcription factors OCT4, SOX2, C-MYC, and KLF4 were kindly provided to us by Dr. Sergei L. Kiselev (Moscow). Three lentiviral constructs (FUW-TRE Ngn2/Puro, containing full-length mouse Ngn2 and puromycin-resistance genes, and M2RtTA and FUW-TRE EGFP, containing the GFP gene) were kindly provided to us by Dr. Thomas Südhof (Stanford, CA, USA). Patient's fibroblasts samples were obtained from the collection «Biobank of populations of Northern Eurasia» financed by the Federal Agency for Scientific Organizations of Russia Federation program for supporting the bioresource collections in 2017 (project no. 0550-2017-0019). We thank the family of the patient with 3p26.3 microduplication for their collaboration. We are grateful to the donors of skin fibroblasts and to their parents for their voluntary participation in the research. Cell culture was performed at the Collective Center of ICG SB RAS "Collection of Pluripotent Human and Mammalian Cell Cultures for Biological and Biomedical Research" (state project No. 0324-2016-0002 of Russian Academy of Sciences).